• Nghien CIFU - Ky thuat
2 Ahire B R . Rane B. R., Bakliwal S. R., Pawar S. P. (2010), "Solubility enhancement of poorly water soluble dmg by solid dispersion techniques", Int J.
Phami Tech. Res. 2(3), pp 2007-2015 3 Anshu S., Jam 0. P (2011). "Solid dispersion:
A promising technique fo enhance solubility of poorly
{Ngdy nhdn bdi: 15/03/2015 - Ngdy duydt ddng: 04/05/2015)
water soluble drug", International Joumal of Drug Delivery, 3, pp 149-170.
4 Lee CR, Fauld D. (1995). "Altretamin. a pharmacokinetic properties, and therapeutic potential m cancer chemotherapy". Drugs. Vol 49. pp. 932 -953
Xay dyng phu'ong phap danh gia anh hyong cua thuoc len enzym cytochrom P450 3A4
thdng qua chuyen hoa nifedipin
Pham Thi Thanh Ha^', Nguygn Thi Thanh Nhai'
' Tnrcmg Dgi hoc Ducrc Hd NQI^ Tnrdng Cao dang Du^rc Trung ucmg Hdi Duang
*E-mail: thanhha.pharm.com
Summary
A method for investigating the inhibition of drugs on CYP3A4 acitivity was developed by following up the metabolism of nifedipine by human CYP3A4 recombinant in the presence and absence of other drugs. Nifedipine was determined by HPLC. V\^th incubation time of 20 minutes and concentration of CYP at 250 nM, over the substrate concentration of 1 to 50 pM, the enzyme kinetic parameters Km and Vmax of nifedipine was found at 6.92 pM and 469 nmol/min/nmol, respectively. Nifedipine was then incubated with CYP3A4 in the presence of 50 nM ketoconazol or 250 pM erythromycin, and Ki was found at 31.4 nM for ketoconazol and 97.5 pM for erythromycin These findings showed the feasibility of the method in evaluating inhibitory effects of drugs on CY3A4 with nifedipine as a substrate.
Keywords: Nifedipine, dehydronifedipine, HPLC, metabolism, CYP3A4, inhibition.
ndng dp co chat trong hdn hgp phan yng chuydn DSt vdn d6
Tyong tdc thudc khflng chi cd dnh hu'fl'ng rdt ign ddn hiflu qua didu tri bflnh md cfl thd gay ra tdc dyng khdng mong mudn nguy hiem. trong dfl, tyong tdc do thay ddi chuydn hfla thudc tgi gan ddng vai tro quan trpng. He enzym cytochrom P450 tham gia chuydn hod hdu hdt cdc thudc tai gan. trong dd. nhirng isoenzym quan trpng nhdt Id CYP1A2, 2B6, 2C9, 2C19, 2D6 va ddc bigt id CYP3A4i^i. Do vdy, viec nghifln ciru dnh hyfl'ng tryc tidp len isoenzym ndy du'gc quan tdm khd nhieu. Phyong phdp nghien cyu anh hyfl'ng Ifln CYP3A4 bdng cdch theo doi mu'c do vd tdc dfl chuydn hod ciia mflt co chat didn hinh qua CYP3A4 ngyoi tai td hgp Id mflt each tiep cdn in vido khd don gidn, chi phi hgp ly. vi chi cdn thidt bj phdn tich thflng thyong de xac djnh
hoa. Vi vdy, phyong phap ndy dygc sir dung khd phd bien tren the gioi'^i; nhyng Igi chya phd bidn fl' Viet Nam. Hau hdt cdc nghien ciru trong nyfl'c sir dung cdch tidp cdn in vivo trfln dgng vdt thi nghiem, tire Id cho dgng vat dimg thudc keo ddi, sau dfl efl lap gan vd djnh lygng CYP trong microsome gani^-^'. Phyong phdp nay khd tdn kem thoi gian va chi phi, hon nira sy cdm yng hay ire che CYP tren dgng vgt chya ehde dd phan anh diing vo'i CYP ciia ngyfl'i. vgi CYP3A4, mgt sd CO chat da dygc sir dyng tir rat ldu nhy erythromycin, nifedipin, midazolam, diazepam, steroids, terfenadin va cyclosporin, trong dd nifedipin, midazolam va testosteron dyge su"
dyng phd bidn nhat. Trong nghien eii'u ndy, Chung toi xay dyng mgt phyong phdp ddnh gid anh hyfl'ng cua thudc len CYP3A4 v6i co chdt
TAP CHI Diroc HOC - 5/2015 (S6 469 NAM 55)
• Nghien CIFU - Ky thuat
nifedipin (NIF). Nifedipin d y g c chuydn hod manh qua CYP3A4 cho san pham chuyen hoa duy nhat Id dehydronifedipin, do do do tin eay tdt'**!. Tinh kha thi ciia phyong phdp d y g c danh gid thdng qua vifle nghien ciru chuyen hod cua nifedipin qua CYP3A4 va so sanh v ^ i cdc sd ligu trong y van, vd sau do, d y g c thir nghiflm u'ng dyng dd ddnh gid dnh hyong eua hai thudc Id ketoconazol vd erythromycin ien CYP3A4.
N g u y i n l i e u v a phu^cyng p h a p Hoa chdt, chdt chudn
Cdc chit chuin
Imipramin.HCI (hdm lygng 99,64 %, chat chudn VKN thudc TW); nifedipin (hdm lyong 99,42 %, Sigma - Aldrich, My); dehydronifedipin (hdm lygng > 98,78 %, Sigma-Aldrich, My)
Hoa chit, dung mdi
Natn hydrophosphat, kali dihydrophosphat, natri hydroxyd, amoni aeetaf, acid phosphoric, acid trifloroacetic, triethylamin dgng tinh khidt phdn tich (Merek, Dye) va methanol, aeetonitril Id dung mdi dimg cho HPLC (Merck, Du-c),
Nguyen lieu khac
NADPH (p-nicotinamid adenin dinucleotid- 2'-phosphat khy, dang mudi tetranatri hydrat)
£ 95 % d y g e mua tir Sigma-Aldrich, Id coenzym cho phdn irng oxy hod NIF. Chd phdm enzym CYP3A4 Supersomes™ (hg baculovirus tdi td hgp mang gen CYP3A4 ngyfl'i, cfl mat ddng thd'i P450 reductase ngu'oi, khong cd cytochrom b5) d y g e mua t y BD Gentest (Woburn, MA, My).
Thidt bj
Hg thdng HPLC Agilent 1200 (Agilent, USA) v&\ detector PDA, cgt sdc ky Cosmosil 5C-18- MS-II (4,6 x 150 mm, 5Mm) (Nacalai. Nhdt).
Phu'O'ng phdp
Phmmg phap 6inh lunyng NIF va DNiF bing HPLC
Nifedipin vd dehydronifedipin d y g c djnh lygng ddng thgi bang HPLC, vo'i phyong phap d y g c nhdm nghien eyu xdy dyng va kiem tra tinh kha thi de djnh lygng trong hdn hgp phdn yng ehuyen hod. Didu kien sde ky nhy sau: Cflt C18 ( 4 , 6 x 1 5 0 mm, 5 pm) 6 nhigt dg phflng; pha dgng methanol-acetonitni-amoniacetat 0 , 0 4 M - tnethylamm 10 % (2,5:1:2:0,05); tdc do dflng
1,2 mL/phut; byfl'c sdng phdt hien 236 nm; ehat chuan ngi imipramin pM; the tieh mau tiflm 20 pL
Chuan bj hdn hap phan dng
- Gdc enzym. CYP3A4 d y g e pha loang vd'i dem phosphat pH 7,4 tgi ndng dp 1 nmol/mL, chia nhfl trong dng Eppendori^ vd bdo quan trong to dd lgnh sdu ( - 80 °C) cho den khi dimg. Khi thye hign phdn irng, enzym d y g c ra ddng trong binh each thuy 37 "C rdi giO lgnh trong n y ^ c da cho ddn khi trfln vol hon hgp phdn yng.
- Gdc CO chdt Dung dich NIF 2 mM d y g c pha trong methanol, rdi pha loang bdng dgm pH 7,4 ddn ndng dfl cdn thidt (gap 4 Idn ndng do d y kien trong hon hgp phdn u'ng).
- Gdc NADPH: Dung djeh 1 mM NADPH trong dem pH 7,4.
- Gdc chuin noi: Dung djch IM1100 pM trong dem pH 7,4 chi diing cho djnh lygng.
- Gdc DNIF: Dung dich DNIF 1 mM pha trong methanol d y o c bdo qudn trong til dd, diing Idm d y o n g ehuan djnh lygng trong ngdy, hodc them vdo mau ehuan NIF de kiem tra phyong phdp hang ngay.
- G6C KETO Dung djch ketoconazol 1 pM trong hdn hgp 1:4 methanol-dflm pH 7.4.
- Gdc ERY: Dung djch erythromycin 2 mM trong hon hgp 1:4 methanol-dgm pH 7,4.
- Ddm phosphat pH 7,4 (dygc dung lam dung mfli pha loang): Dung djeh KH^PO^ 0,02 M, d y g c dieu chmh ddn pH 7,4 bdng dung djch NaOH 0,1 M.
Hdn hgp phan irng d y g c t h y c hien nhy sau' 50 pL dung djch gdc co chat d y g c trgn vd'i 50 pL gdc enzym trong dng Eppendorf 0,5 mL, Idm am 6* 37 "C trong 5 phut. Phan irng d y o c bdt dau khi 50 pL dung dich gdc NADPH d y o c them vao, cimg vol 50 pL dem phosphat (thd tich hdn hgp phan ifng la 200 pL), va d y g c ii fl" 37 °C trong sudt thoi gian phan ipng. Dirng phdn irng bdng cdch thflm 200 pL aeetonitril.
Xd iy miu va djnh Iwqfng
Hdn hgp phan irng sau khi dirng d y g c them 50 pL gdc chudn ngi (ndng dg cudi ciing 10 pM), ldc deu, Igc vd tiem sdc ky. NIF d y g c djnh lygng theo dyfl'ng chuan xay dyng trong ngay tir dung d[ch gdc c o chdt Dung djch DNIF d y g e them vdo dung djeh chuan kiem tra hfl thdng, vd de
TAP CHI Diroc HOC - 5/2015 (S6 469 NAM 55)
Nghien CIFU - Ky thuat
djnh lyong tuy theo yeu cau cua tirng thi nghiflm.
Xdc dinh thong s6 chuyin hoa
Mdi quan he gifra tdc do phan yng tire thoi (V) va ndng dfl eo ehat [S] eo the dygc mfl ta bdng phyong trinh Miehaelis-Menten (a), trong dfl, V^3^ Id tdc do phdn irng tipc thfri tdi da, vd K^
Id hdng sd Michaelis, Id ndng dg co chat fl' % tdc do tdi da (hinh 1). Nghich ddo phyong trinh (a), ta cfl phyong trinh Lineweaver - Bark (b), cho mdi quan he tuyen tinh giira 1/V va 1/[S]. Theo phyong trinh (b), cd thd dd ddng tinh dygc V vd K^ (hinh 1).
V = Vm«[S]
^ m H - [ S l
1 1
AS]
(a) ^ ^ , - —
/A
.— ^ c
1
1/2 V„
1
•
[S]
Hinh 1: Dd thi Miehaelis-Menten (a) vd Lineweaver-Burk (b)
Xdc dfnh hing s6 d-c chiHinh 2 minh hga 3 kidu co chd kim ham (ire chd) dien hinh hay gdp vd'i CYP3A4: kidu (a) egnh tranh (competitive), (b) khflng cgnh tranh (noncompetftive) va (c) phi cgnh tranh
(uncompetitive) NhOng chat cd nhidu hon 1 eo ehd dygc xep vdo nhflm hdn hgp. Cach xac djnh hdng sd ire ehd K, trong moi tryong hgp dygc mfl td trong phyong trinh Lineweaver-Burk tyong irng.
I»> As
€^3 a (^ (A
i« 4 s
q> c^ a
i.^r,H-tiii_L,_L -,i.m,
1 _ K„Hinh 2: C^c W8u ijc chi (a) canh tranh, (b) khdng canh tranh vi (c) phi canh tranh
TAP CHi D l / q C HOC - 5/2015 (SO 469 NAM 55)
Nghien CIFU - Ky thuat
K M qua va ban luan
Nghien c i r u c h u y d n hoa cua nifedipin vo>i CYP3A4
Tuyin tinh theo thai gian phdn tich Chuan bj eac hdn hgp phan irng gdm: 50 pL gdc enzym + 50 pL gdc ca chit ndng dg 100 pM + 50 pL gic NADPH + 50 pL dgm phosphat pH 7.4. Hon hgp phan irng nay chira 25 pM NIF, 0,25 mM NADPH, 250 pmol/mL CYP3A4.
Tien hdnh u cac m i u phdn tich 6' 37*'C trong ede khoang tho'i gian 5 - 1 0 - 1 5 - 2 0 - 3 0 phut; bat hoat enzym bang 200 pL aeetonitril, them 50 pL dung djch gde ehuan nfli, dem djnh lygng ngay.
Tinh todn kdt qua theo d y o n g ehudn NIF vd DNIF d y g c thidt lgp trong ngdy, xdy dyng d y o n g tuyen tinh giii'a ndng do co ehat/san phdm vd thoi gian phdn yng (hinh 3).
Thoi gjan phan irng
^ 2 5 1.20
Tho'i gian (phiit)
Hinh 3; Tuvng quan ndng dd NIF vd DNIF theo th&i gian phdn dng Nhdn xdt: Ket qua eho thdy ndng dfl NIF gidm di vd ndng dfl DNIF tgo thdnh deu tuydn tinh ddn 20 phiJt, va bao hod sau 30 phut, Theo kdt qua ndy, cfl thd lya chgn thd'i gian phan yng tir 10 den 20 phut cho eae nghien cyu chuydn hod tiep theo. Tuy nhidn, ehiing toi chgn thd'i gian ii Id 20 phut, vi mire dg chuyen hod Id cao nhdt se cho phdp thye hign nghien eiru voi ndng do CYP3A4 thdp. tidt klflm chi phi
X&c dinh thdng s6 chuyin hod V^^ vd K„
ctia nifedipin
Di viflc xdc djnh thflng sd Miehaelis-Menten eua NIF ddng tin eay, khoang ndng do co chat khdo sdt it nhat phdi bao phii tir 1/3 K^ ddn 3 K^
v&\ 6 ndng dfl khdc nhau, hoge tdt nhat Id 9-12 ndng dfl tir 1/10 K^ ddn 10 K^ i^i. Chudn bj cdc hdn hop phdn yng gdm: 50 pL gdc enzym + 50 pL gdc CO chdt fl- 9 ndng dg + 50 pL gdc NADPH
+ 50 pL dem phosphat pH 7,4. Nong dg cuoi cilng trong hdn hgp phdn yng Id: 0,25 mM NADPH, 0,25 nmol/mL CYP3A4, vd 1,0-50 pM NIF.
Tidn hdnh u eae mdu phdn tich tren o 37 "C trong thd'i gian 20 phut, bat hogt enzym bang 200 pi aeetonitril, thflm 50 pL gdc chudn nfli IMI Tidn hdnh sdc ki ede mau phdn tieh trfln. Mdi mdu tien hdnh sac ky 3 lan, lay gid trj trung binh.
Tir ndng dg NIF, tinh tdc do phdn yng (bang 1), ve dd thj Miehaelis-Menten (hinh 4). Tinh nghich ddo ndng do NIF 1/[S] va nghjch dao tdc dfl phdn irng 1/V (bang 1).
Do thi Miehaelis-Menten chuyen hoa NIF qua CYP3A4
Hinh 4: Dd thi Miehaelis-Menten cOa NIF ChuySn hod qua CYP3A4 Nhdn xdt Ket qud cho thay ndng dg eo chat bao hod Id > 30 pM, N h y vdy, cdc thong sd dflng hgc ciia CYP3A4 co thd d y g c xdc djnh khi thay ddi khoang ndng do c o ehat < 20 pM. Khoang ndng do NIF cho cdc thi nghiflm tidp theo d y g e ehgn Id 0 - 2 0 pM.
Bdng 1 : Kit qud xdc tfinh tie dd chuyin hda cOa CYP3A4 theo ning dd ISl IVI 1/IS] 1/lVl (liM) (nmot/phul/nmolCYP) diU') (nmol/phul/nmolCYPf
1,0 4,0 5.0 7,5 10 16 20 30 50
0,68 2,02 2,32 2,46 270 3,00 3,14 3,46 3,50 Phi/ong trinh Lineweaver -
Burk
Th6ng so chuyin hoa cua Nl 1,00 0,26 0,20 0,13 0,10 0,067 0,050 0,033 0,020 y=
V ^ = 1,720 0,496 0,431 0,406 0,371 0,333 0,318 0,290 0,285 1,4754xt 0,2134
(r = 0,9952)
4,69 - 6 , 9 2 u M
nmol/phut/nmol
TAP CHi DUOC HOC - 5/2015 (S6 469 NAM 55)
Nghien CJJPU - Ky thuat
Ket qua gia tri K^ = 6,92 pM va V^^, = 4,69 nmol/phut/nmol CYP3A4, nhy vay. Id dat yeu cdu vd khodng ndng dg (1-20 pM t y o n g irng 1/7 K^
den 3 K J . Cdc ket qud nay cung phii hgp vo'i dO* lieu t h y e nghiem trong mflt sd nghien c y u trygd^^i. N h y vgy, eo the diing NIF Idm co chdt dgi dien cho hoat tinh ciia CYP3A4.
Khdo sat dnh hu>o^g cua thudc khac to'i CYP3A4
Sau khi da xdc dmh d y g c thflng sd chuyen
hod ciia NIF vd cdc didu kien tidn hdnh thi nghigm VO'I CYP3A4, Chung tfli irng dyng mfl hinh dd khdo sdt dnh hyfl'ng eua hai thudc dd d y g e biet den Id chdt u-c che CYP3A4, la ketoconazol vd erythromycin"'.
Anh hudng cua ketoconazoi (KET) Tien hdnh phdn ii-ng chuydn hda vd'i ddy ndng dfl 1-20 pM NIF khi co mat 50 nM KET (10 pL gde KET/ 200 pL hfln hgp phdn yng). KET dygc tdch tdt khdi pic can phan tich (hinh 5).
D«)1C,Si9-236,8Re1=oftUJIFEDIPlNEWlFE000038D) iiiAU
300- 250- 2 0 ) - 150- 100- 50-
\
KET
, l \ ^ .A ,s ,:
1 2 3 '
DNIF
TV
5
NIF
y\.
6 7
•
IMI
. s
8 mir Hinh 5: Sdc dd cda mau phdn tich
So sanh tde dfl phan irng tgi tirng thoi diem khi efl va khflng cfl KET, efl s y khdc biet cfl y nghia thdng ke (p = 0,05). Xdy dyng d y o n g eong Lineweaver-Burtt cua NIF khi co mat KET so vol khi khflng cfl KET. Kdt qua trong hinh 6 cho phep xac djnh KET Id loai ire chd khdng eanh tranh, vdi K,= 31,4nM.
Anh hudng cua erythomycin Tidn hdnh phan irng chuydn hoa v&\ day ndng
TAc done cua KETO len chuyen hoa NIF tr 5.0 I
(a)
khi Q 20 pM NIF ciing 50 mM KET dfl 1-20 pM NIF khi co mat 250 pM ERY (10 pL gdc ERY/ 200 pL hon hgp phdn yng). ERY hdp thu UV kem nen khong anh hyfl'ng ddn djnh lygng ciia cdc thdnh phan trong mdu. So sdnh tdc dfl phdn yng tai tirng thdi didm khi cd vd khong efl ERY, cfl s y khde biet efl y nghTa thdng kfl (p = 0,05). Xdy d y n g d y o n g cong Lineweaver- Buri< ciia NIF khi cd mat ERY so vfl'i khi khdng efl ERY. Ket qua trong hinh 6 cho phdp xdc ajnh ERY Id loai ire che cgnh tranh, vol K = 97,5 pM.
c B a
[V] (nmol/pl
-a4
Tac dong ctia ERY len chuyen hoa NIF 6.0
5.0 4.0 3.0 2.0 1.0
"^KTO
(b) / •
• Placebo j ^
• ERY ^ y ^
0 0.2 0.4 0.6 0.8 1.0
1/(S] (nM ») Hinh 6: Anh hud^ng cQa (a) KET vd (b) ERY Idn chuyin hod NIF qua CYP3A4
Ket qud tren hinh 6 cho thay KET la mflt ehat didn hinh, dieu ndy cung thdng nhdt v<^i mflt sd tdi ire chd kieu khdng canh tranh (noneompetrtive) lieu da nghien eyu ve ketoeonazoli*^''. Gid trj K,
TAP CHI Dirac H p c - 5/2015 (S6 469 NAM 55)
• Nghien CLPU - Ky thuat
thu dygc cung phii hgp vfl'i cae gid tri tham khdo trong cdc nghifln ciru tren. Vd'i ERY, co chd dygc xdc djnh la loai (re che canh tranh (competitive), va gid trj Km thu dyge cung phii hgp vol eae tdi lieu tryoc ddyi^^°'.
K i t luan
Mflt phyong phap danh gid anh hyfl'ng cua thudc Ifln CYP3A4 in vitro da dyge xdy dyng.
Phyong phdp ndy dya trfln viflc theo dfli chuyen hod cua NIF, mflt eo chat dien hinh cua CYP3A4.
Chuyen hod eiia NIF ed the dygc theo doi qua sy gidm ndng dfl co chdt NIF hoac qua ndng dfl san phdm ehuyen hod DNIF. Phyong phdp ndy da dyge irng dgng de thir nghiem danh gid kha ndng u'c chd CYP3A4 cua hai ehat Id ketoconazol va erythromycin. Cdc thflng sd chuydn hod K^, V^g, cua NIF cung nhu' he sd u'c che K, eua KET va ERY xdc djnh dygc khd thdng nhdt v^i cdc tdi ligu da cflng bd Tuy loai hinh nghien ciru ehuyen hod in vitro ndy dd dygc irng dung nhieu 6' nyoc ngodi, nhyng 6'Vidt Nam, day Id mgt trong nhCrng nghien cyu dau tien irng dung mfl hinh nay, dae biflt Id vgi ngudn CYP3A4 eLJa microsome gan
ngyoi tai td hgp, mflt ngudn enzym co the cho ket qua dy dodn tdt eho tyong tdc thude in vivo.
Tai lieu tham khao
1 Oao Thj Mai Anh (2003), "Nghien citu anh hu-ong cCia aslem tren he thdng enzym cyt-P450 a gan cua dOng vat thi nghi§m°, Luan van Thac sy du-oc hgc, Trifong Ogi hoc Du-oc H^ NOi.2 D6 Thi Tuyen, Nguyin Thi Ngpc Dao, Nguyin Thai Bidng (2005), Tap chi Sinh hoc. 27 (2); 82-88
3. Galetin A, Clarke SE, Houston JB (2003), Dnig Metab Dtspos. 31" 1108-1116
4. Gibbs MA, Themmel KE, Shen DD, Kunze KL (1999), Drug Metab Dispos , 27(2), 180-187
5 loannides C (2008), "Cytochromes P450; Role in the metabolism and toxicity of drugs and other xenobiotics", RSC Publishing, Cambndge, UK
6 Isoherranen N , Kunze KL, Allen KE., Nelson WL. and Thummel KE (2004), Drug Metab Dispos, 32(10), 1121-1131
7. Ito K , Brown HS. and Houston JB (2004), Br. J Clin Pharmacol-. 57(4), 473-486
8 Kenworthy KE., Bloomer JC , Clarke SE , Houston JB- (1999), Br J Om Pharmacol .A& 716-727
9 PatkiKCVonMoltkeLL.GreenblattDJ (2003).
Drug Metab Dispos.^^ 938-944
10 Zhang X , Jones DR, and Hall SD. (2009), Drug Metab Dispos., 37(1), 150-160
(Ngdy nhdn bdi: 10/03/2015 - Ngdy duydt ddng. 04/05/2015)
V Danh gia tac dung khang khuan cua vien C.T.K. doi vol mot so chung vi khuan
gay viem dwcrng sinh due dwoi
Hoang Thj DiepS Nguyen Tuan Binh' Tran Trgng Dirffng^
'Benh vien Y hpc co fruyen. Bp Cong an
"Cue Y te, Tong cue Hgu cdn - Kp thuat, Bo Cong an
*E-mail: tuanbmh58@gmail. com
Summary
CTK pills vi/ere evaluated for antibacterial activity in vitro against Staphylococcus aureus and Eschenchia coli and some bacterial strains causing inflammation of the lower genital tract isolated from female patients. CTK pills inhibited completely S. aureus ATCC 25923 at the dilution 1/150 and E coli ATCC 25922 at the dilution 1/100. On the S.aureus strains isolated fi-om female patients, CTK pills showed antibacterial activity 100% at the dilution 1/50; 96.7% at the dilution 1/75; 80 % at the dilution 1/100; 63.3 % at the dilution 1/150, and were inactive at the dilution 1/200. CTK pills inhibited the isolated E. coli strains by 90 % at the dilution 1/50; 76.7% at the dihition 1/75; 66.7% at the dilution
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